Tuberculosis is the leading cause of death worldwide from a curable infectious agent and is becoming a major concern due to the spread of drug resistant Mycobacterium tuberculosis (Mtb) strains. In 2015, the South African National Tuberculosis Programme introduced Bedaquiline (BDQ) to strengthen existing regimens for the therapy of rifampicin-resistant TB. This initiative has immediate and far-reaching implications for thousands of South Africans suffering from TB. Concerningly, mutations associated with BDQ resistance (rv0678 and atpE) have been identified in Mtb clones isolated from patients who have never been treated with BDQ or clofazimine (CFZ). Intriguingly, both BDQ and CFZ target the Mtb electron transport chain. These findings, together with the fact that Mtb can persist in a dormant, drug-tolerant state, sometimes reactivating to cause TB decades after the primary infection, indicate an urgent need to better understand the mechanisms of BDQ/CFZ resistance in clinical strains of Mtb. Our long-term goal is to understand the mechanisms of Mtb drug resistance and how this knowledge can be used for prophylactic and therapeutic purposes in South Africa. In this proposal, our central hypothesis is that mutations in rv0678, atpE and elsewhere in the Mtb genome dysregulate central metabolism that contributes to BDQ and CFZ resistance. To test this hypothesis, we have established a global collaborative effort between basic and clinical investigators at Sefako Makgatho Health Sciences University (SMU) in South Africa and the University of Alabama at Birmingham (UAB). As part of this collaboration, we have established a series of specific aims to determine the prevalence of rv0678 and atpE mutations in specific Mtb lineages isolated from patients in South Africa. We will also make use of a novel technology termed extracellular flux (XF96) analysis that we have adapted for studying Mtb bioenergetics in real time. This technology will be complemented by 13C stable isotope analyses using liquid chromatography mass spectrometry. Lastly, we will further pursue our exciting preliminary findings and determine whether BDQ resistance associated variants contribute to the bacilli's bioenergetic flexibility. This contribution is significant, because it has the potential to identify a new paradigm that will lead to a mechanistic understanding of the emergence of BDQ/CFZ resistance, and how disruption of this process could be exploited to sterilize Mtb. This proposal is innovative in our opinion, because the newly adapted technology that is supported by whole genome sequencing, real-time bioenergetics and metabolomics, distinguishes itself from conventional approaches for studying drug resistance in pathogenic microbes.